Establishment of multiplex PCR assay for detection of HIV-1

Abstract

Objective To establish an HIV-1 diagnostic assay using multiplex nested polymerase chain reaction (nPCR) and reverse transcription RT-PCR. Methods Three sets of primers targeting HIV-1 gene of gag, pol and gp41 were designed respectively to establish an nPCR to detect HIV DNA and HIV RNA with RT-PCR. The established multiplex PCR detection system was conducted on 150 HIV seropositive and 50 seronegative patients. The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and reproducibility were calculated with ELISA with results of WB as the gulden standard. Seventy-three positive samples were measured by the established multiplex nPCR and RT-PCR methods, and the sensitivity was compared with HIV-1 nucleic acid sequence based amplification (NASBA) method. The subtypes of 43 positive DNA samples was identified. Results The sensitivities of multiplex nPCR and RT-PCR assays were respectively 98.0% (147/150) and 91.3% (137/150), while the specificities of both methods were 100%. The sensitivity of multiplex nPCR (97.3%) was higher than that of NASBA (72.6%) significantly (χ2=17.34, P=0.01). The sensitivity of multiplex RT-PCR (79.5%) compared with that of NASBA (72.6%), with no significant differences (χ2=0.94, P=0.332). Forty-three DNA samples were identified as 37 subtype B', 5 AE subtype and 1 BC subtype. Conclusions An HIV-1 multiplex nPCR and RT-PCR diagnostic assay system has been developed. The system is easy to operate, economical, highly sensitive and specific, and reproducible, and may cover the major circulating strains in China. Key words: HIV infections; HIV-1; Polymerase chain reaction; Reverse transcriptase polymerase chain reaction